African Journal of Biotechnology Vol. 11(23), pp. 6345-6349, 20 March, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3445 ISSN 1684–5315 © 2012 Academic Journals Full Length Research Paper In vitro anti-hyperglycaemic effect of glucocapparin isolated from the seeds of Boscia senegalensis (Pers.) Lam. ex Poiret Mahamat Nour Adam Sakine 1, Yaya Mahmout 1*, M. G. Dijoux-Franca 2, Joachim Gbenou 3 and Mansourou Moudachirou 3 1Laboratoire de Recherche sur les Substances Naturelles, Faculté des Sciences Exactes et Appliquées, Université de N’djamena, Tchad. 2Département de Botanique, Pharmacognosie et Phytothérapie, UMR CNRS Ecologie Microbienne. Institut des Sciences Pharmaceutiques et Biologique – Faculté de Pharmacie, Université Claude Bernard – Lyon 1, France. 3Laboratoire de Pharmacognosie et des Huiles Essentielles (LAPHE) Université d’Abomey-Calavi, Benin. Accepted 14 February, 2012 Glucocapparin (I) used in this study was isolated from the seeds of Boscia senegalensis (family Capparidaceae). The structure of (I) was determined on the basis of an extensive analysis of the spectroscopic data. Brine shrimp lethality bioassay of (I) showed a marked significant cytotoxic activity with LC 50 = 16.482 µg/ml. Compound (I) reduced the liberation of glucose from the liver of rabbits. The active concentration was 30 mg/ml showing that the in vitro anti-hyperglycaemic effect shown could be related to the traditional use of B. senegalensis seeds in Chad against type 2 diabetes. Key words: Boscia senegalensis , glucocapparin, liver glucose inhibition, cytotoxicity. INTRODUCTION Boscia senegalensis (family Capparidaceae) is a the Peuhls from Senegal, but also an important famine common tree in open scrub or savanna woodland, but food. The fruit is fermented into beer in the Sudan. The can also form a thick understorey in woodland and dry leaves are used to protect stored food against parasites forest. This is a Sahelo-Saharan species which occurs (Hans, 2000). According to the African folk medicine, an below the 20th parallel, while its southern limit runs from infusion of leaves is used to remove intestinal parasites Senegal to northern Burkina Faso, the Niger-Nigerian from camels. Roots are vermifuge and leaves mixed with border, the southern bank of lake Chad and western millet flour taken each morning on an empty stomach is Sudan. It is mainly found under rainfalls of 100 to 300 anthelminthic; dried leaves or dried bark are taken for mm. This plant is an evergreen undershrub or more schistosomiasis. Infusion of the leaves is used as an rarely a shrub, usually 1 to 2 m tall, but sometimes up to eyewash and for pruritus of the eye due to syphilis (Orwa 4 m (Arbonnier, 2002). The fruit is often gathered for et al., 2009; Von Maydell, 1986). In Chad, seeds are human consumption; usually very acidic, it becomes used for the treatment of diabetes. edible after soaking in water for about a week. Fruits are In our previous studies, the hydro alcoholic extracts of frequently sold in the markets for food. It is a regular item the seeds of B. senegalensis have been screened for of diet in Saharo-Sahelian zone and in Sahel, further hyperglycaemia induced by oral administration of D(+)- south, they are mainly supplementary, but their main glucose to the albinos rabbits. With the dose of 250 value is as an emergency food. Seeds are staple food of mg/kg, body weight showed a real anti-hyperglycaemic effect. The seeds were characterized by the presence of alkaloids, saponins, tanins and mucilages (Adam et al., 2011). *Corresponding author. E-mail: [email protected]. Tel: In leafy twigs, L-stachydrine, hydroxy-3 stachydrine, (+235) 66 29 22 10. choline, the sterols β-sitosterol, campesterol, 6346 Afr. J. Biotechnol. stigmasterol, as well as aliphatic alcohols (predominantly transformed into a straight line by means of a trendline fit linear C30, C34, C38), carbohydrates and four glycosinolates regression analysis (MS Excel version 7). The LC 50 was derived were detected. Three glucosinolates were identified as from the best-fit line obtained. The experiment was carried out three times and the mean of the reading was required. methyl, 2-propylisothiocyanate and 2-butylisothiocyanate (Orwa et al., 2009; Bruneton, 1999). In this paper, we reported on the isolation and In vitro study on the hepatic liberation of glucose characterization of glucocapparin (I) by spectroscopic analyses and we evaluated its toxicity and the level of Based on the procedure of the washed liver achieved by Claude hepatic liberation of glucose from the liver of rabbits. Bernard (Grmek, 1997) and used elsewhere (Dods, 2003; Tietz, 1995; Trinder, 1969), a rabbit was sacrificed; the abdominal cavity was incised, the liver was taken out, immediately weighed and dropped in a flask containing the standard physiological solution at MATERIALS AND METHODS pH 7.4 (Mac Ewen solution). Then, the liver was cut into pieces of 300 mg average weight and washed with the Mac Ewen solution. The seeds of B. senegalensis were harvested in November 2008, Each piece was dropped in five different solutions: Solution A: 1 ml around the Northern part of Ndjamena. The species were identified of the Mac Ewen (control); Solution B, C, D, E, F: 1 ml of the Mac and voucher specimen was deposited at the “Laboratoire Ewen + a drop of emulsifier + 10, 20, 30, 40, 50 mg/ml of Vétérinaire et Zootechnique de Farcha” (n° 1344). The air-dried compound (I), respectively. materials were then further dried in an oven at 50°C. Each concentration was tested 5 times. The different solutions were homogenized and incubated for 20 min at room temperature. After this period, 10 µl of each sample were taken out with a Extraction and isolation micropipette and placed in a cell. In this volume, was added 1 ml of the enzymatic reagent whose composition was as follows: The air dried and powdered seed (210 g) of B. senegalensis were phosphate buffer pH 7.4: 13.8 mmol/L, phenol: 10 mmol/L, 4- extracted successively, using a Soxhlet extractor with petroleum aminoantipyrine: 0.3 mmol/L, glucose oxydase: ≥10 000 U/L and ether (750 ml), ethylene trichloride (500 ml) and methanol (750 ml) peroxydase: ≥700 U/L (GLUCOSE PAP SL, SAS, SPPIM, VOIT, with intervals of 12 h between each extraction. The different FRANCE). The whole sample-reagent was then homogenised and extracts were concentrated using rotary evaporator at a maximum incubated for 15 min. The concentrations of glucose at different temperature of 45°C. The weight of each dried crude extract was times, from 0 to 120 min were determined after the enzymatic 8.91, 1.35 and 18.79 g, respectively. The methanolic residue was oxidation by the measurement of the optical density at λ = 500 nm dissolved into a few quantities of the same solvent and refrigerated using a spectrophotometer DREL 2400 HACH. for one day in the freezer. The amorphous crystals obtained were separated from the mother liquid and washed with pure cold methanol. The white substance was re-crystallized from methanol. Data analysis It showed a single spot on TLC precoated silica gel 60 F 254 with the solvent system 15:85 ethyl acetate/hexane. The obtained 2.84 g of crystals were designated as compound I. All the data reported were expressed as mean ±S.E.M. Statistical analysis were performed using Statistica Statsoft V 5.5. The values were considered to be significantly different when the p value was Instrumentation less than 0.05 as compared to the respective control. One and two-dimensional NMR spectra were recorded on a BRUKER AV300 spectrometer. Optimal frequencies were 300 MHz RESULTS AND DISCUSSION for 1H and 75 MHz for 13 C. Mass spectra were recorded with a Thermo LCQ Advantage spectrometer. Melting point was determined in capillaries on Electrothermal ® melting point Chemical characterization apparatus. Compound (I), crystals m.p. 204 to 205°C, yielded 1.35% of the dry material. It showed on thin layer chroma- Brine shrimp cytotoxicity assay tography (TLC) a spot with Rf = 0.52. The negative mode - The brine shrimp lethality biossay was performed as described in ESI mass spectrum showed a peak at 332 [M-H] , Meyer et al. (1982) and McLaughlin (1991). Brine shrimp ( Artemia corresponding to the molecular formula C 8H15 O9S2. It salina Leach) eggs were hatched in a hatching chamber filled with showed also peaks at m/z 664.7, 674 and 687, - + fresh sea water at 28°C, under conditions of continuous illumination corresponding to the fragments [2M-H] , [2M+Na - CH 3] and strong aeration. Separately, 13 mg of compound (I) were and [2M+Na - 2H] +, respectively. 1H and 13 C spectra dissolved in 10 ml of methanol and from these, 130, 65, 32.5, 16.2, exhibited signals from 3 to 5 ppm and at 60 to 105 ppm, 8.1, 4 and 2 µg/ml were prepared by serial dilution. After 48 h, the larvae (nauplii) were collected. A suspension of 16 nauplii in sea respectively which are characteristics of protons and water was added to each sample vial, with a volume of 5 ml and the carbons of a sugar unit. The doublet of the anomeric sample vials were incubated for 24 h at room temperature. After this proton, H-1’, at 4.74 ppm, had a coupling constant of 7.2 period, the numbers of nauplii survivors were counted in each Hz, revealing a β-linked glycoside moiety (Iori et al., sample vial. Larvae were considered dead if they did not exhibit any 2008; Kiddle et al., 2001; Reichelt et al., 2002). The work external movement during several seconds of observation. Pure of carbons and protons, achieved by HMBC and HMQC methanol was used as control.